The long term goal of the proposed research is to determine the cellular and molecular basis for regulation of chloride secretion in epithelia by adenosine receptors. Understanding the autacoid regulation f ion transport by adenosine requires (1) identification of the primary molecular structure and corresponding structure activity profiles of specific adenosine receptor subtypes present in epithelia, (2) identification of the specific ion channels that are the ultimate effectors of adenosine receptors, and (3) integration of the metabolism and transport of adenosine into regulatory schemes of receptor function. Advances in molecular biology of G-protein coupled receptors now provide the basis for a systematic approach to identifying adenosine receptor subtypes. We will combine molecular characterization of unique adenosine receptor subtypes with electrophysiologic characterization of ion channels (C1 and K) and measurements of adenosine release cell membranes to obtain a comprehensive picture of the feedback regulation of chloride secretion by adenosine. Our proposed research focuses on the highly specialized, homogeneous, NaC1 secreting shark rectal gland in which unique adenosine receptor subtypes - A2e and A1e receptors - dually regulate chloride secretion. by exploiting advantages of this model system we propose to provide molecular and functional characterization of these receptors. Specific aims are: (1) to clone, sequence, and functionally express in oocytes and receptor deficient cells the unique A2e and A1e adenosine receptor subtypes that regulate NaC1 transport in the rectal gland; (2) to identify the ion channels (C1 and K) that are coupled to these receptor subtypes and to characterize the pathways of this regulation; and (3) to determine the polarized transport of adenosine across basolateral and apical membranes of rectal gland cells and characterize newly recognized apical receptors for adenosine ATP and UTP. The experiments will employ PCR based cloning and sequencing strategies, measurements of Isc and polarized adenosine release in monolayers of primary cultures and patch clamp studies using cultured rectal gland cells. At each stage of the proposed studies we will correlate molecular structure with functional assays for the receptors.